Oxygen Restriction Generates Difficult-to-Culture P. aeruginosa.

Induction of a non-culturable state has been demonstrated for many bacteria, e.g., Escherichia coli and various Vibrio spp. In a clinical perspective, the lack of growth due to these non-culturable bacteria can have major consequences for the treatment of patients. Here, we show how anoxic conditioning (restriction of molecular oxygen, O2) generates difficult-to-culture (DTC) bacteria during biofilm growth. A significant subpopulation of Pseudomonas aeruginosa entered a DTC state after anoxic conditioning, ranging from 5 to 90% of the total culturable population, in both planktonic and biofilm models. Anoxic conditioning also generated DTC subpopulations of Staphylococcus aureus and Staphylococcus epidermidis (89 and 42% of the total culturable population, respectively). Growth of the DTC populations were achieved by substituting O2 with 10 mM NO3 - as an alternative electron acceptor for anaerobic respiration or, in the case of P. aeruginosa, by adding sodium pyruvate or catalase as scavengers against reactive oxygen species (ROS) during aerobic respiration. An increase in normoxic plating due to addition of catalase suggests the molecule hydrogen peroxide as a possible mechanism for induction of DTC P. aeruginosa. Anoxic conditioning also generated a true viable but non-culturable (VBNC) population of P. aeruginosa that was not resurrected by substituting O2 with NO3 - during anaerobic respiration. These results demonstrate that habituation to an anoxic micro-environment could complicate diagnostic culturing of bacteria, especially in the case of chronic infections where oxygen is restricted due to the host immune response.

Implants induce a new niche for microbiomes.

Although much work is being done to develop new treatments, research and knowledge regarding factors underlying implant-related microbial colonization leading to infection are less comprehensive. Presence of microorganisms in and around implants clinically characterized as uninfected remains unknown. The objective of this study was to detect and identify bacteria and fungi on implants from various groups of patients with no prior indications of implant related infections. Patient samples (implants and tissue) were collected from five different hospitals in the Capital region of Denmark. By in-depth microbiological detection methods, we examined the prevalence of bacteria and fungi on 106 clinically uninfected implants from four patient groups (aseptic loosening, healed fractures, craniofacial complications and recently deceased). Of 106 clinically uninfected implants and 39 negative controls investigated, 66% were colonized by bacteria and 40% were colonized by fungi (p < 0.0001 compared to negative controls). A large number of microbes were found to colonize the implants, however, the most prevalent microbes present were not common aetiological agents of implant infections. The findings indicate that implants provide a distinct niche for microbial colonization. These data have broad implications for medical implant recipients, as well as for supporting the idea that the presence of foreign objects in the body alters the human microbiome by providing new colonization niches.

Tools for studying growth patterns and chemical dynamics of aggregated Pseudomonas aeruginosa exposed to different electron acceptors in an alginate bead model.

In chronic infections, bacterial pathogens typically grow as small dense cell aggregates embedded in a matrix consisting of, e.g., wound bed sludge or lung mucus. Such biofilm growth mode exhibits extreme tolerance towards antibiotics and the immune defence system. The bacterial aggregates are exposed to physiological heterogeneity and O2 limitation due to steep chemical gradients through the matrix, which is are hypothesised to contribute to antibiotic tolerance. Using a novel combination of microsensor and bioimaging analysis, we investigated growth patterns and chemical dynamics of the pathogen Pseudomonas aeruginosa in an alginate bead model, which mimics growth in chronic infections better than traditional biofilm experiments in flow chambers. Growth patterns were strongly affected by electron acceptor availability and the presence of chemical gradients, where the combined presence of O2 and nitrate yielded highest bacterial growth by combined aerobic respiration and denitrification.

The Consequences of Being in an Infectious Biofilm: Microenvironmental Conditions Governing Antibiotic Tolerance.

The main driver behind biofilm research is the desire to understand the mechanisms governing the antibiotic tolerance of biofilm-growing bacteria found in chronic bacterial infections. Rather than genetic traits, several physical and chemical traits of the biofilm have been shown to be attributable to antibiotic tolerance. During infection, bacteria in biofilms exhibit slow growth and a low metabolic state due to O2 limitation imposed by intense O2 consumption of polymorphonuclear leukocytes or metabolically active bacteria in the biofilm periphery. Due to variable O2 availability throughout the infection, pathogen growth can involve aerobic, microaerobic and anaerobic metabolism. This has serious implications for the antibiotic treatment of infections (e.g., in chronic wounds or in the chronic lung infection of cystic fibrosis patients), as antibiotics are usually optimized for aerobic, fast-growing bacteria. This review summarizes knowledge about the links between the microenvironment of biofilms in chronic infections and their tolerance against antibiotics.

Pseudomonas aeruginosa Aggregate Formation in an Alginate Bead Model System Exhibits In Vivo-Like Characteristics.

Alginate beads represent a simple and highly reproducible in vitro model system for diffusion-limited bacterial growth. In this study, alginate beads were inoculated with Pseudomonas aeruginosa and followed for up to 72 h. Confocal microscopy revealed that P. aeruginosa formed dense clusters similar in size to in vivo aggregates observed ex vivo in cystic fibrosis lungs and chronic wounds. Bacterial aggregates primarily grew in the bead periphery and decreased in size and abundance toward the center of the bead. Microsensor measurements showed that the O2 concentration decreased rapidly and reached anoxia ∼100 µm below the alginate bead surface. This gradient was relieved in beads supplemented with NO3- as an alternative electron acceptor allowing for deeper growth into the beads. A comparison of gene expression profiles between planktonic and alginate-encapsulated P. aeruginosa confirmed that the bacteria experienced hypoxic and anoxic growth conditions. Furthermore, alginate-encapsulated P. aeruginosa exhibited a lower respiration rate than the planktonic counterpart and showed a high tolerance toward antibiotics. The inoculation and growth of P. aeruginosa in alginate beads represent a simple and flexible in vivo-like biofilm model system, wherein bacterial growth exhibits central features of in vivo biofilms. This was observed by the formation of small cell aggregates in a secondary matrix with O2-limited growth, which was alleviated by the addition of NO3- as an alternative electron acceptor, and by reduced respiration rates, as well as an enhanced tolerance to antibiotic treatment.IMPORTANCEPseudomonas aeruginosa has been studied intensively for decades due to its involvement in chronic infections, such as cystic fibrosis and chronic wounds, where it forms biofilms. Much research has been dedicated to biofilm formation on surfaces; however, in chronic infections, most biofilms form small aggregates of cells not attached to a surface, but embedded in host material. In this study, bacteria were encapsulated in small alginate beads and formed aggregates similar to what is observed in chronic bacterial infections. Our findings show that aggregates are exposed to steep oxygen gradients, with zones of oxygen depletion, and that nitrate may serve as an alternative to oxygen, enabling growth in oxygen-depleted zones. This is important, as slow growth under low-oxygen conditions may render the bacteria tolerant toward antibiotics. This model provides an alternative to surface biofilm models and adds to the comprehension that biofilms do not depend on a surface for formation.

Antibiotic penetration and bacterial killing in a Pseudomonas aeruginosa biofilm model.

OBJECTIVES: Treating biofilm infections successfully is a challenge. We hypothesized that biofilms may be considered as independent compartments with particular pharmacokinetics. We therefore studied the pharmacokinetics and pharmacodynamics of tobramycin in a seaweed alginate-embedded biofilm model. METHODS: Seaweed alginate beads containing Pseudomonas aeruginosa were cultured in LB medium, sampled at day 1, 3, 5 or 7 and examined for the effect of treatment with tobramycin for 30 min. Treated beads were homogenized and the number of cfu was determined. The antibiotic concentration in the solution of homogenized beads was measured. Finally, beads were examined for live cells by Syto9 staining and for dead cells by propidium iodide staining using a confocal laser scanning microscope. RESULTS: The antibiotic level in each bead was relatively stable (range 30-42 mg/L; MIC = 1.5 mg/L). There were fewer cfu in the tobramycin-treated beads than the non-treated beads (P < 0.016) and bacterial killing was reduced as the culture period increased from 1 to 7 days. Throughout the study period, increasing size and more superficial positioning of the microcolonies within the beads were demonstrated by confocal laser scanning microscopy. More dead cells (measured by propidium iodide staining) were observed in the treated group of beads, which supports the results obtained by culture. CONCLUSIONS: The present study, simulating the clinical pharmacokinetics of tobramycin, demonstrates fast absorption of tobramycin in an in vitro biofilm model. In addition, this model system enables parallel investigation of pharmacokinetics and pharmacodynamics, providing a model for testing new treatment strategies.